This invention relates to modified strains of Vibrio cholerae that are useful, for example, for immunological protection against cholera and other diseases, as well as to plasmids and methods used to make those strains.
Cholera is a diarrheal disease caused by Vibrio cholerae, a gram-negative bacterium. Orally ingested, V. cholerae grows in the upper intestine of man and produces a soluble protein, cholera toxin, which is responsible for the diarrhea observed in the disease. The toxin is composed of two types of subunits, A and B, and the activity of the intact toxin finds its origin in a proteolytic fragment of the A subunit, the A.sub.1 peptide, which is an enzyme that activates the adenylate cyclase system of target cells [Gill (1975) Proc.Nat'l.Acad.Sci. U.S.A. 72: 2064-2068]. The resultant increase in cyclic AMP in intestinal cells causes the diarrhea seen in the disease. The B subunit is nontoxic, although it does bind to the target cell and facilitate the transport of the A.sub.1 peptide through the cell's membrane [Cuatrecasas, Biochem. 12: 3577-3581 (1973)]. Antibodies directed at the B subunit efficiently inactivate the toxin by blocking the binding of the toxin to the cell surface [Id].
Because cholera toxin is an intestinal disease, commercially available killed bacterial and toxoid vaccines have been relatively ineffective in inducing immunity when administered parenterally. Since the disease itself induces immunity, one must conclude that local intestinal immunity mediated by secretory IgA is probably the most important aspect of acquired immunity to cholera. To stimulate local immunity in the intestine, the bacterial antigens must be delivered through the acid and proteolytic barrier of the stomach. For this reason, live, oral cholera vaccines have been proposed.
Mutants of V. cholerae have been isolated which produce no toxin or produce only the B subunit of the toxin [Finklestein et al., (1974) J. Infect. Dis. 129: 117-123; Honda et al., (1979) Proc.Nat'l.Acad.Sci. USA 76: 2052-2056; Mekalanos et al., (1982) Proc.Nat'l.Acad.Sci. USA 79: 151-155]. However, to be an optimum candidate for a live anti-cholera vaccine, a mutant should: 1) be well-characterized and genetically stable (i.e., it should not revert to the toxin-producing wild-type); 2) colonize well in the intestine; and 3) provide long-lived, broad-based immunity. Mutants produced thus far, for example, those produced by chemical mutagenesis or those produced from parent strains not known to have optimal colonization and immunity-producing ability, may pose problems in one of the three areas listed above, even though preliminary testing in human volunteers shows they are relatively innocuous and they induce significant immunity [Woodward et al. (1975), Proc. 11th Jt. Conf. on Cholera NIH, p. 330; Holme et al., Acute Enteric Infections in Children, New Prospects for Treatment and Prevention (1981) Elsevier/North-Holland Biomedical Press, Ch. 26, pp. 443 et seq. (Levine et al.)]